Cell-type-specific translational control of spatial working memory by the cap-binding protein 4EHP

The consolidation of learned information into long-lasting memories requires the strengthening of synaptic connections through de novo protein synthesis. Translation initiation factors play a cardinal role in gating the production of new proteins thereby regulating memory formation. Both positive and negative regulators of translation play a critical role in learning and memory consolidation. The eukaryotic initiation factor 4E (eIF4E) homologous protein (4EHP, encoded by the gene Eif4e2) is a pivotal negative regulator of translation but its role in learning and memory is unknown. To address this gap in knowledge, we generated excitatory (glutamatergic: CaMKIIα-positive) and inhibitory (GABAergic: GAD65-positive) conditional knockout mice for 4EHP, which were analyzed in various behavioral memory tasks. Knockout of 4EHP in Camk2a-expressing neurons (4EHP-cKOexc) did not impact long-term memory in either contextual fear conditioning or Morris water maze tasks. Similarly, long-term contextual fear memory was not altered in Gad2-directed 4EHP knockout mice (4EHP-cKOinh). However, when subjected to a short-term T-maze working memory task, both mouse models exhibited impaired cognition. We therefore tested the hypothesis that de novo protein synthesis plays a direct role in working memory. We discovered that phosphorylation of ribosomal protein S6, a measure of mTORC1 activity, is dramatically reduced in the CA1 hippocampus of 4EHP-cKOexc mice. Consistently, genetic reduction of mTORC1 activity in either excitatory or inhibitory neurons was sufficient to impair working memory. Taken together, these findings indicate that translational control by 4EHP and mTORC1 in both excitatory and inhibitory neurons are necessary for working memory. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-023-00995-2.


Introduction
Experiments in the 1960's provided the first evidence for an essential role of protein synthesis in memory formation [1]. Mice trained in a Y-maze shock avoidance task were unable to recall which arm of the Y-maze was linked to the aversive shock 3 days after bilateral intracerebral injection of the protein synthesis inhibitor, puromycin [1]. However, the use of protein synthesis inhibitors provides only limited insight into the biomolecular processes underlying memory. Genetic animal models served as essential tools towards this end. One of the seminal discoveries first documenting that translational control mechanisms govern memories showed that preventing the phosphorylation of eukaryotic initiation factor (eIF)2α, a molecular brake mechanism for protein synthesis, increased general neural translation and enhanced long-term memory (LTM) [2,3]. mRNA translation is highly regulated at the initiation stage by a heterotrimer protein complex termed eIF4F which interacts directly with the mRNA 5' cap structure (m7GpppN cap, where N is any nucleotide and m is a methyl group). eIF4F is composed of a capbinding protein eIF4E, a molecular scaffolding protein eIF4G, and an mRNA helicase eIF4A [4]. Disrupting the interaction between eIF4E and eIF4G using the inhibitor 4EGI-1 prevents LTM [5]. The eIF4E homologous protein (4EHP or eIF4E2) is also a 5' cap-binding protein, but in contrast to eIF4E it cannot interact with eIF4G and represses translation initiation under most circumstances [6] (hypoxia being an exception [7]). 4EHP inhibits translation via the microRNA (miRNA)induced silencing complex (miRISC) [8,9]. Despite having a weaker cap-binding affinity than eIF4E [10], 4EHP can compete with eIF4E for binding the cap upon recruitment by the 4E transporter protein (4E-T) and miRISC [8]. Importantly, translational control via miRNA-induced mRNA silencing plays a critical role in synaptic plasticity and memory [11]. Previously, we identified a critical role for 4EHP in regulating synaptic plasticity (long-term depression) and mediating social behaviors [12].
Since 4EHP is important for brain function, most likely through miRNA regulation of specific mRNAs important for plasticity-related events at the synapse, we investigated its cell-type-specific activity in learning and memory. To this end, we generated 4EHP conditional knockout (cKO) mice using the Cre-Lox system in excitatory and inhibitory neurons. We did not observe alterations in LTM, but rather an impairment in shortterm working memory (WM). We observed reduced ribosomal protein S6 phosphorylation, a prominent readout of the mechanistic target of rapamycin complex 1 (mTORC1) activity, in CA1 hippocampal pyramidal neurons of 4EHP-cKO exc mice. Consistently, conditional deletion of Raptor, the defining component of mTORC1, resulted in impaired WM. Deletion of 4EHP in GABAergic neurons likewise resulted in impaired WM without affecting LTM. The latter effect was likely due to a developmental abnormality, since the total number of GAD67positive cells was reduced.
Taken together, our findings reveal an important cell-type-specific role of 4EHP in regulating WM possibly through direct modulation of mTORC1 activity, consistent with the known and well-established role of miRNA-mediated regulation of the mTORC1 signaling pathway [13][14][15][16][17], including in the brain [18].

Mice
Adult (i.e. postnatal day [P] 60-90 [19]) male mice on Jackson Laboratory C57BL/6J background were used for experiments. Mice were housed by sex and genotype after weaned at P21 in groups of 2-5 animals per cage. Mice were kept at standard room temperature (RT): 20-22 °C on a 12 h light/dark cycle (7:00-19:00 light period) with food and water access ad libitum. Behavioral experiments were conducted in a soundproof room between 8:00 and 16:00. All behavioral apparatuses were cleaned between animals. Mice were handled 2 times (once per day for 2 days) and habituated in the behavioral room for 20 min prior to behavioral testing. See below for detailed behavioral methods. For data acquisition, analysis and manual scoring, the experimenter was blind to mouse genotype which was randomized throughout the day and across days (in the case of multi-day experiments). Animal care, handling, and experiments were performed according to the guidelines of the Canadian Council on Animal Care and approved by the McGill University Animal Care Committee.

Genotyping
Mouse genotype was determined for each animal using PCR and gel electrophoresis. Eif4e2 gene was amplified using 5'-TCA GAG CAA GAA CAC TTA CAG GAC CAAG forward and 5'-GGC CCA GCC TGC CTG GCA TTC TAG TGG reverse primers. The PCR product was separated through a 1.5% agarose gel using a 150 V potential difference. WT bands were detected around 700 bp and floxed bands at 850 bp. To detect the presence of Cre (300 bp), the forward 5'-GAT TGC TTA TAA CAC CCT GTT ACG and reverse 5'-GTA AAT CAA TCG ATG AGT TGC TTC A primers were used.

Immunofluorescence on brain slices
Mice were placed under general isoflurane anesthesia until loss of pain reflex. Mice were then transcardially perfused with filtered ice-cold PBS then ice-cold 4% PFA. Dissected brains were placed in ice-cold 4% PFA overnight at 4 °C for post-fixation and then moved to 30% sucrose in PBS for 3 d at 4 °C for cryoprotection. 20 µm coronal sections were prepared using a cryostat and adhered to glass coverslips Fisher).

Morris water maze
The Morris water maze (MWM) memory task was performed as previously described [24]. Mice were trained to locate a hidden platform with either 1 trial per day, maximum 60 s per trial (weak) or 3 trials per day, maximum 120 s per trial (strong) over 5 consecutive days in a circular pool 1 m in diameter. Learning was determined manually by timing the latency to locate the hidden (submerged) platform (i.e. escape latency). For probe trials on the following day (day 6), the platform was removed from the maze and the animals were given 60 s to navigate the maze. The percentage of time spent in each quadrant of the maze (quadrant occupancy) was recorded using an automated video tracking system (HVS Image, Buckingham, UK).

T-maze
The T-maze is a test to measure spatial WM. The T-maze consists of a runway (stem) with a left and right arm choice at the end. The maze is enclosed by short walls so the animal can navigate using cues in the environment. Mice were individually placed at the bottom of the stem. For the training phase, one of the maze arms was closed and the animal was allowed to freely explore the other maze arm for 10 min. After 1 h, the mouse was reintroduced to the maze with exception that the animal could enter the previously closed and unexplored arm. We used the animal's innate preference for novelty to probe their ability to alternate exploratory behavior of the previously unencountered T-maze arm. Each trial was recorded using an overhead camera and performance was quantified manually. Time spent in the familiar arm and novel arm were scored. From this data we further calculated discrimination index (DI, see equation below).

Novel object recognition
We assessed working memory using a short-term object recognition test. Mice were first placed inside a white box with an open top measuring 50 × 50 × 30 cm. On day 1, mice were habituated to the empty box once for 10 min. After 24 h, mice were placed back in the box with two identical objects and allowed to explore them for 10 min (training). After 1 h, mice were placed back in the box where one of the two objects were replaced with a novel object. Mice freely explored the objects for 10 min (test). Time spent exploring the familiar object and novel object were scored. From this data we derived the discrimination index (DI, see equation above).

Contextual fear conditioning
Mice were placed in a sound-proof box containing an enclosed isolation chamber with an electric grid floor and overhead camera. Mice were recorded for 2 min before receiving a mild foot shock (0.7 mA, 1 s). After 1 min, mice were removed and placed back in their home cage. 1 or 24 h later, mice were placed back in the enclosure (context) and recorded for 4 min. The average percent freezing over 4 min was used as an assessment of LTM.

Statistical analysis
Statistical analysis was performed on GraphPad Prism 9. An unpaired t-test was used to compare one experimental parameter. Mixed design two-way ANOVA was used to compare two experimental parameters (i.e. genotype as an independent variable and arms in the elevated plus maze test as a repeated measure). Bonferroni test was used for pair-wise post-hoc analysis where there was a significant interaction in the data. A Welch's corrected t-test was used where the difference in variance between groups was significantly different according to the Levene's test. Data were expressed as mean ± s.e.m. and p values < 0.05 were considered statistically significant. Details of all statistics used are listed in Additional file 4: Table S1.

Conditional deletion of 4EHP in CaMKIIα-positive excitatory neurons
We previously generated and studied an excitatory neuron-specific 4EHP KO mouse model which displayed impaired sociability, hyperactivity, and synaptic plasticity dysfunction which is reminiscent of autism spectrum disorder (ASD) [12]. We used the Emx1 promoter to drive Cre expression in excitatory neurons since EMX1 is expressed during embryonic development [25]. Deleting 4EHP early in development was chosen to examine developmental effects which are characteristic of ASD.
Here, we sought to preclude potential developmental effects of 4EHP on learning and memory by driving Cre expression under the Camk2a promoter, which was originally reported to operate at P19 [21]. More recent work has indicated activity at P14 [26,27]. To generate excitatory neuron-specific deletion of 4EHP, we crossed Eif4e2 flx/flx with mice expressing Cre recombinase under the Camk2a promoter (Fig. 1A). Genotyping confirmed homozygous floxed alleles and the presence of Cre (Fig. 1B). Immunofluorescence imaging of the CA1 hippocampus using antibodies against 4EHP and CaM-KII revealed successful cell-type-specific KO of 4EHP directed in excitatory neurons (Fig. 1C). We further confirmed that deletion of 4EHP occurred at P60 (by 22.90 ± 7.28%, Fig. 1D, F) but not at P0 (Fig. 1 D and E) as determined by western blotting. The remaining 4EHP protein in the hippocampus at P60 is possibly due to relatively high levels of expression in other cell types such as glia and inhibitory neurons.

4EHP is dispensable for long-term memory formation
Given the necessity of de novo protein synthesis in LTM formation, we first tested 4EHP-cKO exc mice for longterm contextual fear memory ( Fig. 2A). Mice were placed in a context with an electric grid floor and scored for naïve freezing prior to receiving either a weak (0.3 mA) foot shock for 1 s or a medium strength (0.7 mA) foot shock for 1 s. The weak shock was used to prevent a ceiling effect of freezing behavior and allow discrimination of possible memory enhancement in 4EHP-cKO exc mice. LTM was assessed by quantifying freezing behavior 24 h following the foot shock. Under both conditions, LTM was intact in 4EHP-cKO exc mice (Fig. 2B, C). Next, we subjected mice to the MWM spatial navigation learning and memory task (Fig. 2D). Mice were trained to locate a hidden platform over 5 days having been given a training protocol of either 1 trial per day (weak) or 3 trials per day (strong). With a weak training protocol, mice decreased the time needed to find the platform after 5 days (Fig. 2E), but did not show memory retention on the 6 th day test where the hidden platform was removed, and mouse quadrant occupancy was scored (Fig. 2F). Given a strong training protocol, 4EHP-cKO exc mice had a significantly increased latency to locate the hidden platform on training day 2 by 16 s but were comparable to WT on days 3 to 5 (Fig. 2G). Both WT and 4EHP-cKO exc mice showed Data are presented as mean ± s.e.m. **p < 0.01; ns, not significant. P value calculated using an unpaired t-test. Sample size is located within the bar graph for each group a similar preference for the target quadrant during the test on the following day (Fig. 2H). Importantly, mice significantly decreased the time needed to find the platform during training (Fig. 2E, p = 0.0002; Fig. 2G , p< 0.0001; Additional file 4: Table S1). Together, these data indicate that 4EHP in excitatory neurons does not impact LTM.

Spatial working memory requires 4EHP in excitatory neurons
Working memory (WM), as opposed to LTM, is the short-term storage and manipulation of information for the guidance of decision-making behavior [28]. Deletion of the translational repressor protein 4E-BP2 leads to impairment of WM in mice [29]. This finding suggests that cap-dependent translation controls WM, but the mechanistic details are largely unknown. We tested 4EHP-cKO exc and WT mice in a T-maze spatial WM task (Fig. 3C). Similar to 4E-BP2 KO mice, we observed robust WM impairment in 4EHP-cKO exc mice compared to WT controls (Fig. 3D). Total exploratory behavior, which serves as a control for potential confounding effects of impaired exploratory behavior on memory, was not affected (Fig. 3E). The memory impairments were specific to spatial WM, as short-term (1 h, Fig. 3A) contextual fear memory was not impacted (Fig. 3B). We further validated these findings using an object recognition WM test (Additional file 1: Fig. S1A). Consistent with results from the T-maze, 4EHP-cKO exc demonstrated impaired object recognition WM (Additional file 1: Fig.  S1B) without changes in total exploration (Additional file 1: Fig. S1C). From these data, we conclude that 4EHP in excitatory neurons is necessary for intact spatial WM.

Sustained inhibition of translation impairs working memory
We next sought to elucidate how loss of 4EHP disrupts WM based on its known function in mediating miRNAinduced translational silencing [8]. We previously performed an unbiased ribosome profiling study where the translational efficiency (TE) of mRNAs in mouse embryonic fibroblasts (MEFs) lacking 4EHP expression were determined compared to WT [9]. In this study, the ERK1/2 signalling pathway was negatively regulated by 4EHP deletion via translational upregulation of the ERK1/2 phosphatase Dusp6 [9]. However, we did not previously observe changes in p-ERK1/2 in 4EHP KO brain [12]. Given that miRNAs have been shown to regulate the mTOR pathway activity in the brain [18], we investigated the activity of mTORC1 in the hippocampus of 4EHP-cKO exc mice using immunofluorescence analysis. We observed a 36.40 ± 7.96% reduction in ribosomal protein S6 phosphorylation (which is a readout of mTOR activity) at serine position 240 and 244 [p-S6 (S240/44)] in 4EHP-cKO exc excitatory neurons (Fig. 4A, B), consistent with reduced mTORC1 activity. Furthermore, mice lacking the defining component of the mTORC1 complex (Raptor) in excitatory neurons (Rptor flx/flx :Camk2a-Cre), which results in reduced p-S6 (S240/44) levels (Fig. 4C), have impaired WM (Fig. 4D), without changes in total exploratory behavior (Fig. 4E). Together, these data suggest that WM is impaired because of prolonged attenuation of mTORC1 via loss of 4EHP.

Spatial working memory requires 4EHP and mTORC1 in inhibitory neurons
We next investigated WM in inhibitory neuron-specific 4EHP KO mice (4EHP-cKO inh ). We first validated the deletion of 4EHP in GABAergic (GAD67-positive) neurons by immunofluorescence (Fig. 5A) in the prefrontal cortex where 4EHP is primarily expressed in neuronal cell types (Additional file 2: Fig. S2). Like 4EHP-cKO exc mice, 4EHP-cKO inh also demonstrate WM impairment (Fig. 5B) without changes in total exploratory behavior (Fig. 5C). Long-term contextual fear memory was not affected when 4EHP was deleted in inhibitory neurons (Fig. 5D). We explored the possibility that 4EHP regulates mTORC1 activity in inhibitory neurons by quantifying p-S6 in the GAD67-positive neurons. When normalized to the area of the cell, we did not detect a difference in p-S6 fluorescence signal in 4EHP-cKO inh compared to WT (Fig. 5E, F). However, the total number of GAD67positive cells was significantly reduced (37.82 ± 9.24%) in 4EHP-cKO inh (Fig. 5G). Importantly, mTORC1 activity is still required for WM in inhibitory neurons as Gad2-directed deletion of Raptor (Fig. 5H) impaired WM (Fig. 5I) without affecting total exploratory behavior (Fig. 5J). We conclude that both 4EHP and mTORC1 in inhibitory neurons are required for proper WM.

Discussion
Rapid de novo protein synthesis in neurons is required for forming long-term memories [30]. Temporally inhibiting eIF4E-dependent translation during consolidation prevents LTM formation [31]. Similarly, translational control via phosphorylation of eIF2α on Ser 51 acts as a critical gatekeeper of LTM as the non-phosphorylatable Ser51Ala mutant mice have a lower threshold for L-LTP and enhanced LTM [2,3]. Given the necessity of de novo protein synthesis for LTMs and the emerging importance of microRNAs in synaptic plasticity and memory [11], we investigated the role of 4EHP in LTM. To this end, we subjected 4EHP conditional KO mice to a variety of LTM tests. First, using a contextual fear memory paradigm (CFC), we tested both 4EHP excitatory and inhibitory neuronal KO mice for memory 24 h post-training. In both models, memory retention was comparable to WT controls (Fig. 2B, C and Fig. 5D, respectively). We further assessed 4EHP-cKO exc mice in the MWM where we did not observe any changes to LTM given either a mild or strong training regime (Fig. 2D-H). Given these results, we conclude that 4EHP is not required in excitatory or inhibitory neurons for LTM. Earlier studies documented WM impairments in mice lacking mRNA translation-repressing proteins, such as 4E-BP2 [29] and PERK (an eIF2α kinase) [32,33]. Here, we show that a third translational suppressor, 4EHP is required for WM in both excitatory and inhibitory neurons (Figs. 3D and 5B). Since deletion of 4EHP in either cell type abolishes WM, we conclude that the coordinated function of excitatory and inhibitory neurons mediates WM, as opposed to each cell type independently promoting WM.
We previously showed that mice lacking 4EHP in excitatory neurons defined by EMX1 exhibit impaired preference for social novelty in the 3-chamber sociability assay [12]. It can be argued that this impairment is mediated by a WM deficiency, rather than social interaction. However, 4EHP-EMX1 KO mice were also impaired in the direct/ reciprocal social assay which does not depend on memory retention of a previous social encounter, unlike the Fig. 3 Working memory is impaired in 4EHP-CaMKIIα KO mice. A Schematic depicting contextual fear conditioning (CFC) training and short-term memory test regime. B Short-term contextual fear memory calculated by % freezing 1 h after receiving a mild foot shock (0.3 mA, 1 s) (WT n = 10, cKO exc n = 9). C Schematic depicting T-maze spatial WM test. Mice were placed in a T-maze facing away from the junction (starting point) with one of either arm blocked for 10 min. After 1 h, mice were placed back in the maze with free access to both arms for 10 min of exploration. D T-maze memory is shown as a discrimination index for the novel vs. familiar arm where 0% means equal time spent in both arms (WT n = 11, cKO exc n = 12). E Total exploration in either arm. Data are presented as mean ± s.e.m. ### p < 0.001; ns, not significant. P value calculated using one sample t-test or unpaired t-test. Sample size is located within the bar graph for each group 3-chamber social interaction test. Furthermore, 4EHP-cKO inh mice, which have impaired WM, do not show ASD-like impairments, i.e., social deficits in the 3-chamber assay or exaggerated grooming behavior (Additional file 3: Fig. S3). This raises an intriguing possibility that 4EHP function is not only important for social behavior, but also for cognition in general. This is consistent with findings that executive functions, such as WM, are often impaired in patients with ASD [34][35][36]. Together these studies on 4EHP not only shed light on the mechanistic complexity mediating memory but may offer a potential therapeutic avenue for treating neurodevelopmental disorders such as ASD.
In attempts to understand how 4EHP mediates WM at the cellular and molecular level, we observed a reduction in p-S6 levels in 4EHP null excitatory neurons (Fig. 4A,  B), consistent with attenuated activity of mTORC1 signalling. One possible explanation for this finding is that 4EHP represses the translation of mRNAs which encode for proteins that control this signalling pathway. This type of regulation was previously observed in an unbiased ribosome profiling study where the translational efficiency (TE) of mRNAs in mouse embryonic fibroblasts (MEFs) lacking 4EHP expression were determined compared to WT [9]. In this study, TE of the ERK1/2 phosphatase Dusp6 was significantly upregulated in 4EHP KO MEFs which resulted in reduced p-ERK1/2 levels, impairments in cell proliferation, and increased apoptosis [9]. In our previous study, we did not observe changes in p-ERK1/2 in 4EHP KO brain [12]. Another possibility show same freezing behavior as WT (n = 9) 24 h after receiving the foot shock. E Immunofluorescence analysis of p-S6 (Ser240/244) in 4EHP WT vs. cKO inh . GAD67-positive inhibitory neurons (identified with white arrows) are displayed in red and p-S6 in green. Scale bar represents 60 µm. F Quantification of Mean Gray Value, which is the Integrated Density normalized to area, revealed no difference in p-S6 fluorescence intensity between 4EHP WT (n = 4) and cKO inh (n = 4). G The number of GAD67 cells were counted in 4EHP-cKO inh (n = 4) vs. WT (n = 4). H Immunofluorescence analysis of p-S6 (Ser240/244) in Rptor flx/flx :Gad2-Cre mouse hippocampus compared to Rptor +/+ :Gad2-Cre, scale bar represents 20 µm. I Discrimination index for T-maze working memory in Rptor flx/flx :Gad2-Cre (n = 10) compared to Rptor +/+ :Gad2-Cre (n = 10). J Total exploration time of both maze arms. Data are presented as mean ± s.e.m. **p < 0.01; ns, not significant. P value calculated using an unpaired t-test. ### p < 0.001, calculated using one sample t-test. Sample size is located within the bar graph for each group is that reduced mTORC1 activity in cells lacking 4EHP may be a compensatory mechanism to maintain proteostasis. This would be consistent with our previous results showing that global rates of de novo protein synthesis are not changed in the absence of 4EHP in the brain [12]. However, reduction in mTORC1 activity itself can drive WM impairments, as deletion of Raptor alone engendered impaired WM (Fig. 4C, D). This suggests that mTORC1 activity regulates the expression of critical factors involved in WM. In this case, the WM impairments in 4EHP-cKO exc mice are likely a direct consequence of reduced mTORC1 activity by 4EHP-mediated translational control of factors that regulate the mTORC1 pathway.
We further assessed whether mTORC1 activity in 4EHP-cKO inh mice is dysregulated. We did not observe a difference in p-S6 in GAD67-positive neurons in 4EHP-cKO inh compared to controls (Fig. 5E, F). Instead, the total number of GAD67 cells was reduced in 4EHP-cKO inh mice, indicating a developmental abnormality; this is possibly due to the early expression of Cre in Gad2-Cre mice [22]. These results are consistent with the known role of 4EHP in proper mammalian development, as full-body 4EHP KO mice are born underdeveloped and fail to survive [20]. This phenotype may be underlying the observed WM impairment, but further research is required to establish a causal relationship. Deletion of Raptor in inhibitory neurons was sufficient to impair WM (Fig. 5H, I). Together, our results suggest that 4EHP and sustained mTORC1 activity in excitatory or inhibitory neurons is necessary to support WM. Future research is required to determine whether mTORC1 is directly modulated by 4EHP and miRNA-mediated translational control to mediate WM. These mechanistic studies will ideally be carried out in multiple brain regions which are important for WM, such as the hippocampus [37] and prefrontal cortex [38].

Limitations
It is necessary to understand how 4EHP mechanistically regulates mTORC1 activity. The hypothesis that 4EHP controls the translation of kinases and/or phosphatases in the mTORC1 pathway could be studied using an unbiased mRNA translation analysis approach, such as Translating Ribosome Affinity Purification (TRAP) [39] or RiboTag, according to our previous work [3]. In these methods, a tagged ribosomal protein is expressed through an adeno-associated virus (AAV) under Cre recombinase control. After purifying and sequencing ribosome-bound mRNAs, we can evaluate ribosome occupancy on mRNAs as a measure of active translation across genotypes [40]. This would enable the analysis of the mRNAs in cell types that are translationally regulated by 4EHP.